Systematic evaluation of methods for iron-impregnation of biochar and effects on arsenic in flooded soils.

There is a need for innovative strategies to decrease the mobility of metal(loids) including arsenic (As) and cadmium (Cd) in agricultural soils, including rice paddies, so as to minimize dietary exposure to these toxic elements. Iron (Fe)-modified biochars (FBCs) are used to immobilize As and Cd in soil-water systems, but there is a lack of clarity on optimal methods for preparing FBCs because there are only limited studies that directly compare BCs impregnated with Fe under different conditions. There is also a lack of information on the long-term performance of FBCs in flooded soil environments, where reductive dissolution of Fe (oxy)hydroxide phases loaded onto biochar surfaces may decrease the effectiveness of FBCs. This study uses material characterization methods including FTIR, SEM-EDX, BET, and adsorption isotherm experiments to investigate the effects of Fe-impregnation methods (pH, pyrolysis sequence, and sonication) on the morphology and mineralogy of Fe loaded onto the biochar surface, and to FBC adsorbent properties for arsenate (As(V)), arsenite (As(III)), and Cd. Acidic impregnation conditions favored the adsorption of As(III) onto amorphous Fe phases that were evenly distributed on the biochar surface, including within the biochar pore structure. The combination of sonication with acidic Fe-impregnation conditions led to the best adsorption capacities for As(V) and As(III) (4830 and 11,166 µg As g-1 biochar, respectively). Alkaline Fe-impregnation conditions led to the highest Cd adsorption capacity of 3054 µg Cd g-1 biochar, but had poor effectiveness as an As adsorbent. Amending soil with 5% (w/w) of an acid-impregnated and sonicated FBC was more effective than an alkaline-impregnated FBC or ferrihydrite in decreasing porewater As concentrations. The acid-impregnated FBC also had greater longevity, decreasing As by 54% and 56% in two flooded phases, probably due to the greater stability of Fe(III) within the biochar pore structure that may have a direct chemical bond to the biochar surface. This study demonstrates that FBCs can be designed with selectivity towards different As species or Cd and that they can maintain their effectiveness under anaerobic soil conditions. This is the first study to systematically test how impregnation conditions affect the stability of FBCs in soils under multiple drying-rewetting cycles.

FOXL2 interaction with different binding partners regulates the dynamics of ovarian development.

The transcription factor FOXL2 is required in ovarian somatic cells for female fertility. Differential timing of Foxl2 deletion, in embryonic versus adult mouse ovary, leads to distinctive outcomes, suggesting different roles across development. Here, we comprehensively investigated FOXL2's role through a multi-omics approach to characterize gene expression dynamics and chromatin accessibility changes, coupled with genome-wide identification of FOXL2 targets and on-chromatin interacting partners in somatic cells across ovarian development. We found that FOXL2 regulates more targets postnatally, through interaction with factors regulating primordial follicle formation and steroidogenesis. Deletion of one interactor, ubiquitin-specific protease 7 (Usp7), results in impairment of somatic cell differentiation, germ cell nest breakdown, and ovarian development, leading to sterility. Our datasets constitute a comprehensive resource for exploration of the molecular mechanisms of ovarian development and causes of female infertility.

Characteristics and Outcomes of Postgraduate Midwifery Fellowships: A Mixed Methods Study.

INTRODUCTION: Postgraduate fellowships are growing in midwifery and yet are poorly understood by the profession. These fellowships are optional for midwives interested in developing advanced skills, transitioning to specialty practice, or entering or re-entering professional practice. The purpose of this study was to explore the characteristics and outcomes of US postgraduate midwifery fellowships as described by midwifery fellowship program directors. METHODS: Postgraduate midwifery fellowship programs in the United States were identified through an environmental scan and snowball sampling. Directors of these fellowship programs were invited to complete a survey and an open-ended interview to determine their program's characteristics and outcomes. Quantitative data were summarized, and thematic analysis was used to explore the qualitative data. RESULTS: An environmental scan and snowball sampling identified 17 postgraduate midwifery fellowship programs. Thirteen fellowship program directors completed a survey (76.5%), and 11 completed an interview (64.7%). The findings identified program characteristics, including location, structure, funding, and educational offerings, of midwifery fellowship programs. The thematic analysis revealed the emergence of 3 themes highlighting the creation of support for midwives in transition, including new graduates and practicing midwives returning to practice after a hiatus or shifting to specialty practice: (1) stepping stones, (2) empowered and equipped, and (3) innovative future. Community birth practice was defined as specialty practice by these directors. DISCUSSION: The results of this study inform the profession of the availability of fellowships and the development of future high-quality midwifery fellowships. Postgraduate midwifery fellowships should not be required for entry to practice but have a place in the work-study opportunities available to US midwives.

Donor template delivery by recombinant adeno-associated virus for the production of knock-in mice.

BACKGROUND: The ability of recombinant adeno-associated virus to transduce preimplantation mouse embryos has led to the use of this delivery method for the production of genetically altered knock-in mice via CRISPR-Cas9. The potential exists for this method to simplify the production and extend the types of alleles that can be generated directly in the zygote, obviating the need for manipulations of the mouse genome via the embryonic stem cell route. RESULTS: We present the production data from a total of 13 genetically altered knock-in mouse models generated using CRISPR-Cas9 electroporation of zygotes and delivery of donor repair templates via transduction with recombinant adeno-associated virus. We explore the efficiency of gene targeting at a total of 12 independent genetic loci and explore the effects of allele complexity and introduce strategies for efficient identification of founder animals. In addition, we investigate the reliability of germline transmission of the engineered allele from founder mice generated using this methodology. By comparing our production data against genetically altered knock-in mice generated via gene targeting in embryonic stem cells and their microinjection into blastocysts, we assess the animal cost of the two methods. CONCLUSIONS: Our results confirm that recombinant adeno-associated virus transduction of zygotes provides a robust and effective delivery route for donor templates for the production of knock-in mice, across a range of insertion sizes (0.9-4.7 kb). We find that the animal cost of this method is considerably less than generating knock-in models via embryonic stem cells and thus constitutes a considerable 3Rs reduction.

Development of novel ß2-adrenergic receptor agonists for the stimulation of glucose uptake - The importance of chirality and ring size of cyclic amines.

ß2-Adrenergic receptor (ß2AR) agonists have been reported to stimulate glucose uptake (GU) by skeletal muscle cells and are therefore highly interesting as a possible treatment for type 2 diabetes (T2D). The chirality of compounds often has a great impact on the activity of ß2AR agonists, although this has thus far not been investigated for GU. Here we report the GU for a selection of synthesized acyclic and cyclic ß-hydroxy-3-fluorophenethylamines. For the N-butyl and the N-(2-pentyl) compounds, the (R) and (R,R) (3d and 7e) stereoisomers induced the highest GU. When the compounds contained a saturated nitrogen containing 4- to 7-membered heterocycle, the (R,R,R) enantiomer of the azetidine (8a) and the pyrrolidine (9a) had the highest activity. Altogether, these results provide pivotal information for designing novel ß2AR agonist for the treatment of T2D.

SARS-CoV-2 detection by a clinical diagnostic RT-LAMP assay.

The ongoing pandemic of SARS-CoV-2 calls for rapid and cost-effective methods to accurately identify infected individuals. The vast majority of patient samples is assessed for viral RNA presence by RT-qPCR. Our biomedical research institute, in collaboration between partner hospitals and an accredited clinical diagnostic laboratory, established a diagnostic testing pipeline that has reported on more than 252,000 RT-qPCR results since its commencement at the beginning of April 2020. However, due to ongoing demand and competition for critical resources, alternative testing strategies were sought. In this work, we present a clinically-validated procedure for high-throughput SARS-CoV-2 detection by RT-LAMP in 25 minutes that is robust, reliable, repeatable, sensitive, specific, and inexpensive.

Treatment with a ß-2-adrenoceptor agonist stimulates glucose uptake in skeletal muscle and improves glucose homeostasis, insulin resistance and hepatic steatosis in mice with diet-induced obesity.

AIMS/HYPOTHESIS: Chronic stimulation of ß2-adrenoceptors, opposite to acute treatment, was reported to reduce blood glucose levels, as well as to improve glucose and insulin tolerance in rodent models of diabetes by essentially unknown mechanisms. We recently described a novel pathway that mediates glucose uptake in skeletal muscle cells via stimulation of ß2-adrenoceptors. In the current study we further explored the potential therapeutic relevance of ß2-adrenoceptor stimulation to improve glucose homeostasis and the mechanisms responsible for the effect. METHODS: C57Bl/6N mice with diet-induced obesity were treated both acutely and for up to 42 days with a wide range of clenbuterol dosages and treatment durations. Glucose homeostasis was assessed by glucose tolerance test. We also measured in vivo glucose uptake in skeletal muscle, insulin sensitivity by insulin tolerance test, plasma insulin levels, hepatic lipids and glycogen. RESULTS: Consistent with previous findings, acute clenbuterol administration increased blood glucose and insulin levels. However, already after 4 days of treatment, beneficial effects of clenbuterol were manifested in glucose homeostasis (32% improvement of glucose tolerance after 4 days of treatment, p < 0.01) and these effects persisted up to 42 days of treatment. These favourable metabolic effects could be achieved with doses as low as 0.025 mg kg-1 day-1 (40 times lower than previously studied). Mechanistically, these effects were not due to increased insulin levels, but clenbuterol enhanced glucose uptake in skeletal muscle in vivo both acutely in lean mice (by 64%, p < 0.001) as well as during chronic treatment in diet-induced obese mice (by 74%, p < 0.001). Notably, prolonged treatment with low-dose clenbuterol improved whole-body insulin sensitivity (glucose disposal rate after insulin injection increased up to 1.38 ± 0.31%/min in comparison with 0.15 ± 0.36%/min in control mice, p < 0.05) and drastically reduced hepatic steatosis (by 40%, p < 0.01) and glycogen (by 23%, p < 0.05). CONCLUSIONS/INTERPRETATION: Clenbuterol improved glucose tolerance after 4 days of treatment and these effects were maintained for up to 42 days. Effects were achieved with doses in a clinically relevant microgram range. Mechanistically, prolonged treatment with a low dose of clenbuterol improved glucose homeostasis in insulin resistant mice, most likely by stimulating glucose uptake in skeletal muscle and improving whole-body insulin sensitivity as well as by reducing hepatic lipids and glycogen. We conclude that selective ß2-adrenergic agonists might be an attractive potential treatment for type 2 diabetes. This remains to be confirmed in humans. Graphical abstract.

Acute ß-adrenoceptor mediated glucose clearance in brown adipose tissue; a distinct pathway independent of functional insulin signaling.

OBJECTIVE: ß-adrenoceptor mediated activation of brown adipose tissue (BAT) has been associated with improvements in metabolic health in models of type 2 diabetes and obesity due to its unique ability to increase whole body energy expenditure, and rate of glucose and free fatty acid disposal. While the thermogenic arm of this phenomenon has been studied in great detail, the underlying mechanisms involved in ß-adrenoceptor mediated glucose uptake in BAT are relatively understudied. As ß-adrenoceptor agonist administration results in increased hepatic gluconeogenesis that can consequently result in secondary pancreatic insulin release, there is uncertainty regarding the importance of insulin and the subsequent activation of its downstream effectors in mediating ß-adrenoceptor stimulated glucose uptake in BAT. Therefore, in this study, we made an effort to discriminate between the two pathways and address whether the insulin signaling pathway is dispensable for the effects of ß-adrenoceptor activation on glucose uptake in BAT. METHODS: Using a specific inhibitor of phosphoinositide 3-kinase α (PI3Kα), which effectively inhibits the insulin signaling pathway, we examined the effects of various ß-adrenoceptor agonists, including the physiological endogenous agonist norepinephrine on glucose uptake and respiration in mouse brown adipocytes in vitro and on glucose clearance in mice in vivo. RESULTS: PI3Kα inhibition in mouse primary brown adipocytes in vitro, did not inhibit ß-adrenoceptor stimulated glucose uptake, GLUT1 synthesis, GLUT1 translocation or respiration. Furthermore, ß-adrenoceptor mediated glucose clearance in vivo did not require insulin or Akt activation but was attenuated upon administration of a ß3-adrenoceptor antagonist. CONCLUSIONS: We conclude that the ß-adrenergic pathway is still functionally intact upon the inhibition of PI3Kα, showing that the activation of downstream insulin effectors is not required for the acute effects of ß-adrenoceptor agonists on glucose homeostasis or thermogenesis.

BRL37344 stimulates GLUT4 translocation and glucose uptake in skeletal muscle via ß2-adrenoceptors without causing classical receptor desensitization.

The type 2 diabetes epidemic makes it important to find insulin-independent ways to improve glucose homeostasis. This study examines the mechanisms activated by a dual ß2-/ß3-adrenoceptor agonist, BRL37344, to increase glucose uptake in skeletal muscle and its effects on glucose homeostasis in vivo. We measured the effect of BRL37344 on glucose uptake, glucose transporter 4 (GLUT4) translocation, cAMP levels, ß2-adrenoceptor desensitization, ß-arrestin recruitment, Akt, AMPK, and mammalian target of rapamycin (mTOR) phosphorylation using L6 skeletal muscle cells as a model. We further tested the ability of BRL37344 to modulate skeletal muscle glucose metabolism in animal models (glucose tolerance tests and in vivo and ex vivo skeletal muscle glucose uptake). In L6 cells, BRL37344 increased GLUT4 translocation and glucose uptake only by activation of ß2-adrenoceptors, with a similar potency and efficacy to that of the nonselective ß-adrenoceptor agonist isoprenaline, despite being a partial agonist with respect to cAMP generation. GLUT4 translocation occurred independently of Akt and AMPK phosphorylation but was dependent on mTORC2. Furthermore, in contrast to isoprenaline, BRL37344 did not promote agonist-mediated desensitization and failed to recruit ß-arrestin1/2 to the ß2-adrenoceptor. In conclusion, BRL37344 improved glucose tolerance and increased glucose uptake into skeletal muscle in vivo and ex vivo through a ß2-adrenoceptor-mediated mechanism independently of Akt. BRL37344 was a partial agonist with respect to cAMP, but a full agonist for glucose uptake, and importantly did not cause classical receptor desensitization or internalization of the receptor.

ß3-Adrenergically induced glucose uptake in brown adipose tissue is independent of UCP1 presence or activity: Mediation through the mTOR pathway.

OBJECTIVE: Today, the presence and activity of brown adipose tissue (BAT) in adult humans is generally equated with the induced accumulation of [2-18F]2-fluoro-2-deoxy-d-glucose ([18F]FDG) in adipose tissues, as investigated by positron emission tomography (PET) scanning. In reality, PET-FDG is currently the only method available for in vivo quantification of BAT activity in adult humans. The underlying assumption is that the glucose uptake reflects the thermogenic activity of the tissue. METHODS: To examine this basic assumption, we here followed [18F]FDG uptake by PET and by tissue [3H]-2-deoxy-d-glucose uptake in wildtype and UCP1(-/-) mice, i.e. in mice that do or do not possess the unique thermogenic and calorie-consuming ability of BAT. RESULTS: Unexpectedly, we found that ß3-adrenergically induced (by CL-316,243) glucose uptake was UCP1-independent. Thus, whereas PET-FDG scans adequately reflect glucose uptake, this acute glucose uptake is not secondary to thermogenesis but is governed by an independent cellular signalling, here demonstrated to be mediated via the previously described KU-0063794-sensitive mTOR pathway. CONCLUSIONS: Thus, PET-FDG scans do not exclusively reveal active BAT deposits but rather any tissue possessing an adrenergically-mediated glucose uptake pathway. In contrast, we found that the marked glucose uptake-ameliorating effect of prolonged ß3-adrenergic treatment was UCP1 dependent. Thus, therapeutically, UCP1 activity is required for any anti-diabetic effect of BAT activation.

Glucose uptake in brown fat cells is dependent on mTOR complex 2-promoted GLUT1 translocation.

Brown adipose tissue is the primary site for thermogenesis and can consume, in addition to free fatty acids, a very high amount of glucose from the blood, which can both acutely and chronically affect glucose homeostasis. Here, we show that mechanistic target of rapamycin (mTOR) complex 2 has a novel role in ß3-adrenoceptor-stimulated glucose uptake in brown adipose tissue. We show that ß3-adrenoceptors stimulate glucose uptake in brown adipose tissue via a signaling pathway that is comprised of two different parts: one part dependent on cAMP-mediated increases in GLUT1 transcription and de novo synthesis of GLUT1 and another part dependent on mTOR complex 2-stimulated translocation of newly synthesized GLUT1 to the plasma membrane, leading to increased glucose uptake. Both parts are essential for ß3-adrenoceptor-stimulated glucose uptake. Importantly, the effect of ß3-adrenoceptor on mTOR complex 2 is independent of the classical insulin-phosphoinositide 3-kinase-Akt pathway, highlighting a novel mechanism of mTOR complex 2 activation.

Improving type 2 diabetes through a distinct adrenergic signaling pathway involving mTORC2 that mediates glucose uptake in skeletal muscle.

There is an increasing worldwide epidemic of type 2 diabetes that poses major health problems. We have identified a novel physiological system that increases glucose uptake in skeletal muscle but not in white adipocytes. Activation of this system improves glucose tolerance in Goto-Kakizaki rats or mice fed a high-fat diet, which are established models for type 2 diabetes. The pathway involves activation of ß2-adrenoceptors that increase cAMP levels and activate cAMP-dependent protein kinase, which phosphorylates mammalian target of rapamycin complex 2 (mTORC2) at S2481. The active mTORC2 causes translocation of GLUT4 to the plasma membrane and glucose uptake without the involvement of Akt or AS160. Stimulation of glucose uptake into skeletal muscle after activation of the sympathetic nervous system is likely to be of high physiological relevance because mTORC2 activation was observed at the cellular, tissue, and whole-animal level in rodent and human systems. This signaling pathway provides new opportunities for the treatment of type 2 diabetes.

Does the emergency medical treatment and labor act affect emergency department use?

BACKGROUND: Emergency departments (EDs) face increasing patient volumes and economic pressures. These problems have been attributed to the Emergency Medical Treatment and Labor Act (EMTALA). STUDY OBJECTIVE: To determine whether modifying EMTALA might reduce ED use. METHODS: We surveyed ED patients to assess their knowledge of hospitals' obligations to treat all patients regardless of insurance and to determine whether knowledge is associated with ED use. RESULTS: Among 4136 study subjects, 72% reported awareness of the law. Sixty-one percent of subjects were moderate ED users (≥ 1 additional ED visit in 12 months). Moderate users more often knew the law (74% vs. 70%, p=0.005). Multivariate regression showed that factors associated with moderate use were: awareness of EMTALA (odds ratio [OR] 1.44; 95% confidence interval [CI] 1.24-1.67), adult patient (OR 1.94; 95% CI 1.69-2.22), and government insurance (OR 2.67; 95% CI 2.30-3.08) or uninsured (OR 1.72; 95% CI 1.42-2.08). Only 8% of subjects were high-frequency users (≥5 visits). High-frequency users were more often aware of EMTALA (78% vs. 72%, p=0.02). Covariates associated with high frequency were EMTALA awareness (OR 1.69; 95% CI 1.28-2.24), adult patient (OR 2.59; 95% CI 2.00-3.36), and government insurance (OR 3.73; 95% CI 2.76-5.06) or uninsured (OR 3.77; 95% CI 2.65-5.35). CONCLUSION: Many patients know that the law requires hospitals to provide care. This knowledge is associated with more frequent ED use. EMTALA changes might reduce ED use, but broader policy implications should be considered.

Neurotoxic and neurotrophic roles of proNGF and the receptor sortilin in the adult and ageing nervous system.

The precursor form of the nerve growth factor (proNGF), forms a heterotrimeric complex with the receptors p75 and sortilin; this complex has been implicated in neuron cell death. However, it is not known whether proNGF and the receptors p75 and sortilin contribute to age- and disease-related neurodegeneration. Here we show that proNGF induces cell death in subpopulations of basal forebrain and peripheral sympathetic neurons of old, but not of young, adult rodents. In contrast, proNGF appears to induce neurite outgrowth rather than cell death of young adult sympathetic neurons. We have examined the neurotoxic role of proNGF in old age, and find that proNGF protein is elevated during ageing in the projection areas of some populations of vulnerable central and peripheral neurons; caloric restriction, which has known neuroprotective effects, partially prevents these increases. Sortilin was found to play a significant part in the observed patterns of age-related proNGF-mediated neurotoxicity. In particular, survival of aged neurons was rescued by neurotensin, an alternative sortilin ligand that blocks the sortilin-mediated effects of proNGF. Furthermore, sortilin immunoreactivity increases markedly in ageing rodent basal forebrain and sympathetic neurons; in contrast, p75 levels are either unchanged or reduced. From these data we propose that selective age-related neuronal atrophy and neurodegeneration may be mediated by increased sortilin expression in neurons, together with elevated levels of proNGF expression in some targets.